Microscopy with application of the so-called total internal reflection fluorescence (TIRF) is a special form of fluorescence microscopy. It is, for example, disclosed in WO 2006/127692 A2 (for example, in FIGS. 9 and 10C). FIG. 1 clarifies the context. The fluorophores F0 of the specimen P are excited to fluoresce F1 by means of an evanescent illumination field E solely in a thin layer behind the interface between the cover glass D and the specimen. The evanescent illumination field E is generated in the specimen, in which the excitation radiation T inside the cover glass D is conducted at an angle θc, which leads to total internal reflection, onto the interface between the cover glass and the specimen. Since only the thin layer is excited to fluoresce, it is possible to attain a very high axial resolution. The optical axial resolution of a TIRF microscope results from the penetration depth d of the evanescent field in the specimen. Depending on the angle of incidence θ, the axial solution is the product ofd=λ/[4π√(n12 sin2 θ−n22)],where λ is the excitation wavelength, n1 is the index of refraction of the cover glass, and n2 is the index of refraction of the specimen medium.
Usually the illumination passes, as shown in FIG. 2 in schematic form, through the microscope objective lens O into its edge region in such a way that on leaving the objective lens O the illumination light crosses the optical axis of the objective lens O at an angle that is greater than or equal to the angle of total internal reflection θc. The microscope objective lens O has to possess a high numerical aperture in order to provide the mandatory large angle of incidence required for the excitation light T. The resulting fluorescence is collected through the same objective lens O and projected onto a CCD camera (not illustrated).